Fig 5.
Activation of PKA via cAMP elevation rescued impairment of proteasome activity and reduced mutant Huntingtin (Htt) aggregates. (A) ST14A cells were transfected with pcDNA3.1-Htt-(Q)25-hrGFP or pcDNA3.1-Htt-(Q)109-hrGFP for 68 h and then treated with the desired reagent(s) (FK, 10 μM; db-cAMP, 100 μM; H89, 10 μM) for another 4 h. Chymotrypsin-like activity of proteasomes was assessed as described in Materials and Methods. Data were normalized to that of control Q25-expressing cells and are presented as the means ± SE for 3 experiments. ∗, P < 0.05 (by one-way ANOVA). (B) ST14A cells were transfected with pcDNA3.1-Htt-(Q)25-hrGFP or pcDNA3.1-Htt-(Q)109-hrGFP for 24 h and then treated with the desired reagent(s) (CGS, 10 μM; CSC, 20 μM; H89, 10 μM; MG132, 1 μM) for another 48 h. Chymotrypsin-like activity of proteasomes was assessed as described in Materials and Methods. Data were normalized to that for control Q25-expressing cells and are presented as the means ± SE for 3 experiments. ∗, P < 0.05 (by one-way ANOVA). (C) ST14A cells were transfected with pcDNA3.1-Htt-(Q)25-hrGFP or pcDNA3.1-Htt-(Q)109-hrGFP for 24 h and then treated with the desired reagent(s) for another 48 h. Lysates (30 μg per lane) collected from the indicated condition were subjected to a Western blot analysis using an antiubiquitin antibody. (D) ST14A cell lysates (30 μg per lane) collected from the indicated condition were subjected to a filter retardation assay. Insoluble Htt aggregates retained on the filter were detected using an anti-Htt antibody. The level of actin in the lysates was assessed using a Western blot analysis and served as an internal control. Representative images of 3 independent experiments are shown. (E) Primary striatal neurons were prepared from E18 R6/2 or wild-type WT mice. Striatal neurons were cotransfected with Snap25-DsRed and Snap25-pZsProSensor-1 at 7 days in vitro (DIV) and then treated with vehicle or 10 μM CGS for 72 h. Representative images of 3 independent experiments are shown. Scale bar = 20 μm.